Chimeric antigen receptors and methods of use

ABSTRACT

The invention relates to a chimeric antigen-receptor polypeptide heterodimer comprising two polypeptides, wherein the first contains an extracellular part of the major histocompatibility complex I alpha chain and the second contains a 32-microglobulin domain, or the first contains an extracellular part of the major histocompatibility complex II alpha chain and the second contains a major histocompatibility complex II beta chain. One of the polypeptides further contains a transmembrane domain, a hinge region and an intracellular domain of the T cell receptor alpha chain and the other one contains a transmembrane domain, a hinge region and an intracellular domain of the T cell receptor beta chain, and additionally an antigen-peptide covalently linked to said extracellular MHC domain. The invention further relates to a method for the identification of a TCR recognizable peptide sequence making use of the heterodimer of the invention.

Most T-cells express αβT-cell receptors (TCRs) and recognize antigens inthe form of peptides (epitopes) presented by the majorhistocompatibility complex (MHC) on other cells. The TCRs of cytotoxicT-lymphocytes recognise epitopes displayed by MHC class I molecules onthe surface of almost every cell in the body. The TCRs of helper T cellsrecognise epitopes displayed by MHC class II molecules on the surface ofantigen-presenting immune cells, including macrophages, dendritic cellsand B-cells. Efficient recognition of the epitope by T cells involvesadditional T cell surface glycoproteins: CD8 on cytotoxic T-lymphocytes(CD8⁺ T cells), and CD4 on helper T cells (CD4⁺ T cells), which bind MHCclass I and II molecules, respectively. The binding of a TCR to anepitope can result in signals being sent to the T lymphocyte's nucleusto induce a T cell response.

Unambiguous and efficient identification of antigenic specificities of Tcells holds great promise for the development of efficient immunetherapies and diagnostic tools. In particular:

-   -   it may help assessing the safety of adoptive T cell therapies,    -   it may enable new approaches in the treatment of autoimmune        diseases, cancer, and in the development of new vaccines.

However, TCRs bind MHC-peptide complexes with low affinity, which makesphage- and yeast-display methods inefficient. Alternative methods, likescreening of positional scanning combinatorial peptide libraries, takeadvantage of the cross-reactivity of the TCR and use peptide pools todefine motifs that lead to T-cell activation. Apart from similaraffinity constrains, these cumbersome methods suffer from a high rate offalse-positive results. Because random peptide sequences areinterrogated, the identified peptide motifs are ambiguous or have noclear homology to native proteins.

The problem underlying the present invention is to provide the means fordirect and sensitive, unbiased identification of antigenic peptidespecificities of CD4⁺ and CD8⁺ T cells for use in vitro and in vivo.This problem is solved by the subject-matter of the independent claims.

Terms and Definitions

Amino acid sequences are given from amino to carboxyl terminus. Capitalletters for sequence positions refer to L-amino acids in the one-lettercode (Stryer, Biochemistry, 3^(rd) ed. p. 21). Lower case letters foramino acid sequence positions refer to the corresponding D- or(2R)-amino acids.

In the context of the present invention, the terms identity or sequenceidentity are used in their meaning known in the art of genetics andbioinformatics; they refer to a single quantitative parameterrepresenting the result of a sequence comparison position by position.Methods of sequence comparison are known in the art; the BLAST algorithmavailable publicly is an example.

One such example for comparison of amino acid sequences is the BLASTPalgorithm that uses default settings such as: Expect threshold: 10; Wordsize: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs:Existence 11, Extension 1; Compositional adjustments: Conditionalcompositional score matrix adjustment. In the absence of furtherdetails, these settings are used for determination of amino acidsequence identity values given below.

One such example for comparison of nucleic acid sequences is the BLASTNalgorithm that uses the default settings: Expect threshold: 10; Wordsize: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2;Gap costs: Linear. In the absence of further details, these settings areused for determination of nucleic acid sequence identity values givenbelow.

In the context of the present specification, the term majorhistocompatibility complex (MHC) is used in its meaning known in the artof cell biology and biochemistry; it refers to a cell surface moleculethat displays a peptide, a fraction of a protein, in a way suitable forthe recognition by a T cell receptor. Peptides recognised by the immunesystem are referred to as epitopes or antigenic peptides oroligopeptides in the context of the present specification.

There are two major classes of MHC molecules: class I and class II.

MHC class I occurs as α chain composed of three domains—α1, α2, α3. Theα3 domain interacts with the non-MHC molecule β2-microglobulin. Thepeptide being displayed or presented is held by the peptide-bindinggroove, in the central region of the α1/α2 heterodimer. The α3 subunitcontains a transmembrane domain, anchoring the MHC class I molecule tothe cell membrane.

MHC class II is formed of two chains, α and β, each having twodomains—α1 and α2 and β1 and β2, respectively. The peptide-bindinggroove (the structural element formed by the MHC class II molecule thatpresents or displays the peptide epitope) is formed by the heterodimerof α1 and β1. The α2 and β2 subunits contain transmembrane domainsanchoring the MHC class II molecule to the cell membrane.

MHC class I and class II molecules comprise in their immature form asignal peptide like the majority of newly synthesized proteins that aredestined towards the secretory pathway. The MHC signal peptide sequenceis located upstream (5′) of the MHC α1 domain on the MHC mRNA molecule.After cleavage of the signal peptide the MHC molecule is referred to asthe mature MHC molecule.

In the context of the present specification, the term β2-microglobulindomain is used in its meaning known in the art of cell biology andbiochemistry; it refers to a non-MHC molecule that is part of the MHCclass I heterodimer molecule. In other words, it constitutes the β chainof the MHC class I heterodimer.

In the context of the present specification, the term T cell receptor(TCR) is used in its meaning known in the art of cell biology andbiochemistry; it refers to a molecule found on the surface of T cellsthat is able to recognize antigens bound to major histocompatibilitycomplex molecules. TCRs are disulfide-linked membrane-anchoredheterodimers consisting of highly variable α and β chains. Each chain iscomposed of two extracellular domains: Variable (V) region and aConstant (C) region. The Variable region binds to the peptide-MHCcomplex; the Constant region is proximal to the cell membrane, followedby a hinge region, transmembrane region and a short cytoplasmic tail.

In the context of the present specification, the term T cell receptorcomplex is used in its meaning known in the art of cell biology andimmunology; it refers to an octameric complex of the heterodimericTCRα/β, with two heterodimeric signaling modules CD3ε/δ and CD3γ/ε andthe homodimer CD247ζ/ζ (also known as TCR ζ-chain or zeta-chain).Ionizable residues in the transmembrane domain of each subunit form apolar network of interactions that hold the complex together. Since thecytoplasmic tail of the TCRα/β is extremely short, making it unlikely toparticipate in signaling, these signaling molecules are vital inpropagating the signal from the triggered TCR into the cell. The mostcommon mechanism for activation and regulation of molecules beneath theplasma membrane is via phosphorylation/dephosphorylation by proteinkinases. The intracellular parts of CD3 and CD247ζ containimmunoreceptor tyrosine-based activation motifs (ITAMs) that aretargeted by the Src family of tyrosine-kinases.

In the context of the present specification, the term functionallylinked refers to the linkage of the activity status of two differentfunctions. For example a receptor polypeptide (function 1) may befunctionally linked to a reporter gene and its promoter (function 2);then, if the receptor changes its activity status (e.g. activated), thepromoter of the reporter gene will also change its activity status (e.g.activated) and the reporter gene is transcribed. One such non-limitingexample is the nuclear factor of activated T cells (NFAT) signalingpathway known in the art. Activation of native TCRs (function 1) in Tcells results in the activation of protein kinases and phosphatases thatinitiate nuclear import of the transcription factor NFAT leading to theexpression of NFAT target genes (function 2). In other words the TCR isfunctionally linked to the expression of NFAT target genes.

In the context of the present specification, the term activation of thereporter gene refers to a change in the activity status of the reportergene. One example of such a change in activity status is the activationof the promoter of a reporter gene. This results in the increasedtranscription/translation of the reporter gene. Another example is thecleavage of an inhibitor of the reporter protein which results in anincreased amount of active reporter protein.

In the context of the present specification, the term transgenic is usedin its meaning known in the art of cell biology; it refers to theintroduction of a exogenous nucleic acid sequence into a living organismso that this organism displays a new property it does not possessendogenously.

In the context of the present specification, the term antigen receptoris used in its meaning known in the art of cell biology and immunology;it refers to surface receptors able to bind antigens or epitopes.Examples for antigen receptors are B-cell and T-cell receptors.

In the context of the present specification, the term chimeric antigenreceptor refers to artificial engineered receptors comprising parts ofantigen receptors. A non-limiting example is a hybrid receptor thatcomprise domains from T-cell or B-cell receptors fused to MHC domainsand/or antigenic peptide sequences.

In the context of the present specification, the term oligopeptide oroligopeptide sequence exclusively refers to T-cell reactiveoligopeptides that, when presented in the context of an MHC molecule ona cell, may elicit a T-cell response if recognized by a cognate T cellreceptor. Where mention is made of an oligopeptide sequence beingcomprised in a (longer) polypeptide of the invention, the skilled personwill understand that this refers to an oligopeptide sequence that can berecognized by a TCR when presented on an MHC molecule. The oligopeptidesequence comprises, in addition to the MHC-presented T cell reactiveepitope, a linker of several amino acids, allowing the T cell reactiveepitope to fit into the MHC molecule. Examples for linker length andsequence are given below.

According to a first aspect of the invention, a method for theidentification of a TCR recognizable peptide sequence is provided,comprising the steps of:

-   -   i. Providing a plurality of mammalian cells, wherein        -   each of the plurality of mammalian cells expresses a member            of a library,        -   each member of the library encodes a transgenic antigen            receptor molecule,        -   the transgenic antigen receptor molecule comprises            -   an extracellular domain of MHC1 or MHC2,            -   an oligopeptide comprised within the polypeptide                sequence of the extracellular domain sequence (the term                within is meant to signify that the oligopeptide can be                comprised anywhere between the N and C boundary of the                extracellular domain sequence, or at any of its                respective ends), wherein the oligopeptide is different                for each member of the library, and wherein the                oligopeptide is presented in a way suitable for the                recognition by a T cell receptor,            -   a transmembrane domain of a T-cell receptor (TCR), and            -   an intracellular domain of a TCR,        -   the transgenic antigen receptor molecule is functionally            linked to a reporter protein, whereby binding of a cognate T            cell receptor to the transgenic antigen receptor results in            the activation of the reporter gene.    -   ii. Contacting the plurality of mammalian cells with a        preparation of T-lymphocytes able to bind to the oligopeptide        sequence comprised in the transgenic antigen receptor through a        T cell receptor expressed on the surface of the T lymphocytes.    -   iii. Separating cells with a detectable reporter protein from        said plurality of mammalian cells according to the level of        detectable reporter protein, thereby yielding activated cells.    -   iv. Isolating DNA encoding the expressed library member from the        activated cells.    -   v. Sequencing of the oligopeptide sequence comprised in the        transgenic antigen-receptor expressed in the activated cells.        The determined sequence of the oligopeptide is the TCR        recognizable peptide sequence.

In other words, the method of the invention allows assessing theantigenic potential of oligopeptides, or—to be more precise—thepotential of a particular oligopeptide sequence to be a MHC-presentedepitope triggering a cognate TCR response. To accomplish thisassessment, a library of transgenic antigen receptors comprisingpotentially antigenic oligopeptides is required. A member of the libraryis expressed in each mammalian cell of the plurality of cells, whereinthe oligopeptide sequence can be different for each member of thelibrary. The oligopeptide is presented by the transgenic antigenreceptor in a way that is suitable for the recognition by a T cellreceptor. Binding of the antigenic oligopeptide by the provided Tlymphocytes via their T cell receptor activates the reporter proteinthat is functionally linked to the transgenic antigen receptor. Themammalian cell is then separated according to the level of detectablereporter. DNA is isolated from the separated mammalian cell and thecomprised oligopeptide DNA is sequenced. Every oligopeptide retrieved bythis method is recognized by a T cell receptor.

The transgenic antigen receptor comprises an oligopeptide that ispresented in a way suitable for the recognition by a T cell receptor, anextracellular domain of MHC1 or MHC2, a transmembrane domain of a T cellreceptor and an intracellular domain of a T cell receptor. Theoligopeptide and the different domains are covalently linked to form asingle polypeptide chain (extracellular-transmembrane-intracelluar). Incertain embodiments, the transgenic receptor additionally comprises ahinge region of a T cell receptor that is situated between theextracellular domain and the transmembrane domain.

In the context of the present specification, the terms expresses orexpression are used in their meaning in the art of cell biology andmolecular biology; they refer to the transcription and translation of aDNA sequence and the derived mRNA.

In certain embodiments the separation of cells with a detectablereporter protein from the plurality of mammalian cells according to thelevel of detectable reporter protein as described in step iii. isperformed at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. Thisembodiment is particularly advantageous if libraries of high complexityare used.

According to an alternative aspect of the invention, a method for theidentification of a TCR recognizable peptide sequence is provided. Themethod comprises:

-   -   i. Providing a mammalian cell, wherein        -   the mammalian cell expresses a transgenic antigen receptor            molecule;        -   the transgenic antigen receptor molecule comprises            -   an extracellular domain of MHC1 or MHC2 comprising an                oligopeptide presented in a way suitable for the                recognition by a T cell receptor,            -   a transmembrane domain of a T-cell receptor (TCR), and            -   an intracellular domain of a TCR,        -   the transgenic antigen receptor molecule is functionally            linked to a reporter protein, whereby binding of a T cell            receptor to the transgenic antigen receptor results in the            activation of said reporter gene.    -   ii. Contacting the mammalian cell with a preparation of        T-lymphocytes able to bind to the oligopeptide comprised in the        transgenic antigen receptor through a T cell receptor expressed        on the surface of the T lymphocyte. Binding of the T cell        receptor to the transgenic antigen receptor activates the        reporter gene, yielding an activated mammalian cell.    -   iii. Isolating DNA encoding the expressed library member from        the activated mammalian cell.    -   iv. Sequencing of the oligopeptide sequence comprised in the        transgenic antigen-receptor.

In certain embodiments the transgenic antigen receptor additionallycomprises a hinge region.

In certain embodiments according to the first and second aspect of theinvention the transgenic antigen receptor is a transgenic antigenreceptor already known in the art.

Examples of suitable transgenic antigen receptors known in the art aredisclosed in Jyothi et al., Nat Biotechnol 20, 1215-1220 (2002); Geigeret al., Blood 98(8) 2364-2371 (2001); US2008286312A1; Mekala et al.,PNAS 102(33), 11817-11822, (2005); Moisini et al., J Immunol (2008),180:3601-3611; Scott et al., J Autimm 35, 390-397 (2010).

In certain embodiments of any of the above aspects of the invention, thetransgenic antigen receptor is a chimeric antigen-receptor polypeptideheterodimer comprising a first polypeptide and a second polypeptide, and

-   -   a. the first polypeptide comprises an extracellular part of the        major histocompatibility complex I (MHC class I) alpha chain and        the second polypeptide comprises a β2-microglobulin domain, or    -   b. the first polypeptide comprises an extracellular part of the        major histocompatibility complex II (MHC class II) alpha chain        and the second polypeptide comprises an extracellular part of        the major histocompatibility complex II (MHC class II) beta        chain.

At least one of the first and the second polypeptide chain additionallycomprises an oligopeptide, covalently linked to the extracellular MHCdomain, wherein the oligopeptide can be recognized by a T cell receptor.

In certain embodiments, the oligopeptide sequence further comprises alinker sequence 4 to 16 amino acids, particularly 6, 8, 10, 12 or 14amino acids in length. In certain embodiments, the linker ispredominantly composed of glycine and one of serine, threonine andalanine. In certain embodiments, the linker comprises glycine and serineonly.

One of the first polypeptide and the second polypeptide furthercomprises a hinge region, a transmembrane domain and an intracellulardomain or intracellular tail of the T cell receptor alpha chain and theother one of the first polypeptide and the second polypeptide comprisesa hinge region, a transmembrane domain and an intracellular domain ofthe T cell receptor beta chain.

In certain embodiments the oligopeptide sequence and a glycine-serinelinker are inserted between the last amino acid of the MHC signal/leaderpeptide and the first amino acid of the MHC α1 domain or β1 domain.

In certain embodiments the oligopeptide sequence and a linker sequenceis inserted after amino acid 1, 2, 3, 4 or 5 of the MHC α1 domainsequence or β1 domain sequence.

Attaching the peptide to the beta chain (β1 domain) will insert thepeptide into the MHC in the most commonly found direction. Attaching itto the alpha chain will insert the peptide in a reverse orientation.

In certain embodiments the extracellular part of the MHC molecule isselected from the human major histocompatibility complex gene family HLAavailable to the person skilled in the art in specialist databases suchas IMGT/HLA (http://www.ebi.ac.uk/ipd/imgt/hla/). Alternatively, the HLAsequence is derived from the RNA extracted from patient blood or tissuesamples.

In certain embodiments the oligopeptide is 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30 or 40 amino acids (AA) in length. Themajority of MHC class I peptides are 8-10 amino acids long whereas MHCclass II can present much longer peptides, as the binding cleft is open,nevertheless the actual epitope ranges from 10 to 12 AA. In the hereindisclosed method the peptides might be even longer than 20 (see FIG. 3,the LCMV peptide found by the method of invention comprises 24 AA). Incertain embodiments, larger oligopeptides are employed, as this allowsscreening of more binding registers on one peptide.

In certain embodiments the hinge region, the transmembrane andintracellular part of the transgenic antigen-receptor in the firstand/or second polypeptide is not derived from the same TCR chain. Inother words the hinge region or the transmembrane domain of the TCRα-chain could be connected to the transmembrane or intracellular domainof the TCR β-chain and vice versa.

In certain embodiments the chimeric antigen-receptor polypeptideheterodimer according to the second aspect of the invention, comprises afirst polypeptide with an amino acid sequence having at least ≥80%, 85%,90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO 007 and asecond polypeptide with an amino acid sequence having at least ≥80%,85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO 008.

According to a third aspect of the invention a chimeric antigen-receptorpolypeptide heterodimer is provided comprising a first polypeptide and asecond polypeptide. The first polypeptide is linked to the secondpolypeptide by one or several disulfide bonds, and

-   -   a. the first polypeptide comprises an extracellular part of the        major histocompatibility complex I (MHC class I) alpha chain and        the second polypeptide comprises a major histocompatibility        complex I (MHC class I) associated β2-microglobulin domain, or    -   b. the first polypeptide comprises an extracellular part of the        major histocompatibility complex II (MHC class II) alpha chain        and the second polypeptide comprises a major histocompatibility        complex II (MHC class II) beta chain.

One of the first polypeptide and the second polypeptide furthercomprises a hinge region, transmembrane domain (and the intracellulardomain or tail) of the T cell receptor (TCR) alpha chain and the otherone of the first polypeptide and the second polypeptide comprises ahinge region, transmembrane domain (and the intracellular domain ortail) of the T cell receptor beta chain.

In other words the chimeric antigen-receptor polypeptide heterodimer isable to present an oligopeptide (epitope) in an MHC-context, in orderfor the oligopeptide (epitope) to be recognized and bound by its cognateTCR. The binding of its cognate TCR results in the activation of theintracellular domains of the CD3 and CD247 molecules associated with thechimeric antigen-receptor polypeptide heterodimer, which leads to theactivation of NFAT.

In certain embodiments, the extracellular parts of the MHC moleculecomprise

-   -   i. MHC class I alpha1 and alpha2 and alpha3 domains on the first        polypeptide (in addition to β-2 microglobulin as the second        polypeptide), or    -   ii. MHC class II alpha1 and alpha2 domains on the first        polypeptide, and MHC class II beta1 and beta2 domains on the        second polypeptide.

In certain embodiments, the first polypeptide comprises substantiallythe entire extracellular part of the major histocompatibility complex I(MHC class I) alpha chain. In certain embodiments, the first polypeptideis the extracellular part of the major histocompatibility complex I (MHCclass I) alpha chain.

In certain embodiments, the second polypeptide comprises substantiallythe entire extracellular part of the major histocompatibility complex I(MHC class I) associated β2-microglobulin domain. In certainembodiments, the second polypeptide is the major histocompatibilitycomplex I (MHC class I) associated β2-microglobulin domain.

In certain other embodiments, the first polypeptide comprisessubstantially the entire extracellular part of the majorhistocompatibility complex II (MHC class II) alpha chain. In certainembodiments, the first polypeptide is the extracellular part of themajor histocompatibility complex II (MHC class II) alpha chain. Incertain embodiments, the second polypeptide comprises substantially theentire major histocompatibility complex II (MHC class II) beta chain. Incertain embodiments, the second polypeptide is the majorhistocompatibility complex II (MHC class II) beta chain.

In certain embodiments at least one of the first and the secondpolypeptide chain additionally comprises an antigen-peptide covalentlylinked to the extracellular MHC domain, wherein said oligopeptide can berecognized by a T cell receptor.

In certain embodiments the antigen-peptide sequence and a glycine-serinelinker are inserted between the last amino acid of the MHC signal/leaderpeptide and the first amino acid of the MHC α1 domain or β1 domain.

In certain embodiments the antigen-peptide sequence and a linkersequence is inserted after amino acid 1, 2, 3, 4 or 5 of the MHC α1domain sequence or β1 domain sequence.

In certain embodiments the extracellular part of the MHC molecule isselected from the human major histocompatibility complex gene family HLAavailable in specialist databases such as IMGT/HLA(http://www.ebi.ac.uk/ipd/imgt/hla/). Alternatively, the HLA sequence isderived from the RNA extracted from patient blood or tissue samples.

In certain embodiments the oligopeptide is 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30 or 40 amino acids in length. The majority ofMHC class I peptides are 8-10 amino acids long whereas MHC class II canpresent much longer peptides, as the binding cleft is open, neverthelessthe actual epitope is inside 10-12 AA. In the herein disclosed methodthe peptides might be even longer than 20 (see FIG. 3, the LCMV peptidefound by the method of invention comprises 24 AA). It could beadvantageous to have larger oligopeptides as this allows screening ofmore binding registers on one peptide.

In certain embodiments the hinge region, the transmembrane andintracellular part of the chimeric antigen-receptor polypeptide in thefirst and/or second polypeptide is not derived from the same TCR chain.In other words the hinge region or the transmembrane domain of the TCRα-chain could be connected to the transmembrane or intracellular domainof the TCR β-chain and vice versa.

According to a fourth aspect of the invention a nucleic acid moleculeencoding the chimeric antigen-receptor polypeptide heterodimer accordingto the third aspect of the invention is provided, particularly a nucleicacid molecule having a promoter sequence operable in a mammalian hostcell.

According to a fifth aspect of the invention a cell, particularly amammalian cell, comprising or expressing the nucleic acid moleculeaccording to the fourth aspect of the invention is provided.

According to a sixth aspect of the invention a cell, particularly amammalian cell, more particularly a mammalian T-lymphocyte is provided,comprising

-   -   i. the chimeric antigen-receptor polypeptide according to the        first aspect of the invention, and    -   ii. an effector function functionally linked to the chimeric        antigen-receptor polypeptide heterodimer.

In certain embodiments the effector function is:

-   -   i. a reporter protein, and/or    -   ii. the ability to induce cell death, particularly apoptosis, in        cells bound to the chimeric antigen-receptor polypeptide        heterodimer.

In certain embodiments the reporter protein is selected from:

-   -   i. a fluorescent protein,    -   ii. a luciferase protein,    -   iii. an antibiotic resistance gene,    -   iv. a Cre recombinase,    -   v. a CAS-9 nuclease, or    -   vi. a CAS-9 chimeric transcriptional suppressor or activator.

According to a seventh aspect of the invention a method for obtaining apreparation of T-lymphocytes having a reduced reactivity against anantigen is provided. The method comprises:

-   -   i. providing a preparation of T-lymphocytes obtained from a        patient,    -   ii. contacting the preparation of T-lymphocytes with mammalian        cells according to the fifth or sixth aspect of the invention,        wherein the mammalian cell is characterized in that the effector        function is able to induce cell death, particularly apoptosis,        in cells bound to the chimeric antigen-receptor polypeptide        heterodimer.

In other words, specific T lymphocytes able to bind to the oligopeptidesequences provided within the chimeric antigen-receptor polypeptideheterodimer of the mammalian cell would undergo cell death, particularlyapoptosis.

According to an eighth aspect of the invention a method for depletion ofT lymphocytes having an activity against specific antigens for use in apatient in need thereof is provided. The method comprises the steps of:

-   -   i. providing a preparation of mammalian cells according to the        fourth aspect of the invention,    -   ii. administering of the mammalian cells to a patient,        wherein the mammalian cell is characterized in that the effector        function is able to induce cell death, particularly apoptosis,        of cells bound to the chimeric antigen-receptor polypeptide        heterodimer.

In other words T lymphocytes able to bind to the oligopeptide sequencethat is displayed in the chimeric antigen-receptor polypeptideheterodimer are selectively depleted. This can be used for example forthe reduction or removal of autoreactive T lymphocytes in a patient withneed thereof.

In certain embodiments this method is used to treat autoimmune diseasessuch as allergies. In certain embodiments this method is used for theprevention and treatment of organ rejection after transplantation.

According to a ninth aspect of the invention, a mammalian cell accordingto the fourth aspect of the invention is provided for use in thetreatment or prevention of autoimmune disease, transplant rejection orimmune dysfunction including but not limited to, type 1 diabetes,multiple sclerosis, rheumatoid arthritis, Crohn's disease andinflammatory bowel syndrome. In other words, the mammalian cell is usedfor the depletion of T-lymphocytes having an activity against specific,disease associated antigens.

According to a tenth aspect of the invention a method for detecting apatient's immune response to an oligopeptide is provided. The methodcomprises the steps of:

-   -   i. providing the mammalian cell according to the fourth aspect        of the invention, wherein said cell comprises a reporter protein        functionally linked to said chimeric antigen-receptor        polypeptide heterodimer,    -   ii. providing a blood sample of the patient ex-vivo containing T        lymphocytes involved in the immune response,    -   iii. contacting the mammalian cell with the blood sample under        conditions allowing for the effector function to operate, and    -   iv. detecting the reporter protein in the mammalian cell.

In other words if in the blood of an individual T lymphocytes able tobind to the used oligopeptide are enriched resulting in a strongreporter protein expression this indicates an immune response againstthe oligopeptide.

In certain embodiments the oligopeptide used in this method is derivedfrom a virus, a bacterium, a fungus or a parasite.

In certain embodiments the method according to the ninth aspect of theinvention further comprises the steps of

-   -   i. separating the mammalian cells according to the expression of        the reporter protein,    -   ii. isolating DNA from the separated mammalian cells, and    -   iii. sequencing of the oligopeptide, encoded in the chimeric        antigen-receptor polypeptide heterodimer according to the first        aspect of the invention.

In other words a multitude of the mammalian cells according to thefourth aspect of the invention with different oligopeptides in thechimeric antigen-receptor polypeptide heterodimer is used. Each of theseoligopeptides is derived from a certain pathogen, allergen, tumour orinflamed tissue (in the case of an autoimmune patient). This allowssimultaneous testing for a multitude of different immune responses. Onenon-limiting example is the use as a diagnostic tool.

Wherever alternatives for single separable features such as, forexample, a linker sequence, linker length, are laid out herein as“embodiments”, it is to be understood that such alternatives may becombined freely to form discrete embodiments of the invention disclosedherein. The person skilled in the art understands that isolated featuresof the invention mentioned as specific embodiments may be combined withany other features mentioned.

The invention is further illustrated by the following examples andfigures, from which further embodiments and advantages can be drawn.These examples are meant to illustrate the invention but not to limitits scope.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1 shows structure and antigen-specific reactivity of the MCR2sensor (chimeric antigen-receptor polypeptide heterodimer withextracellular parts from MHC II). (a) Schematic representation of theMCR2 and its interaction with the peptide-specific TCR. (b) Surfaceexpression of the MCR2(gp61) on BEKO thymoma cells (c) Time course ofMCR2(gp61) (left panel) or MCR2(OVA) (right panel) down-regulation inBEKO cells co-cultured with gp61-specific Smarta or OVA-specific OT-IITCR transgenic CD4+T-cells. Values show MCR2 levels depicted aspercentage of mean fluorescence intensity at the start of co-culture.(d) Time course of peptide-specific NFAT activation (GFP expression) inH18.3.13 cells transduced with MCR2(gp61) or MCR2(OVA) co-cultured withSmarta or OT-II CD4+T-cells. Histogram shows examples of NFAT activationmeasurements in MCR2(gp61)+ cells at indicated time points. (e)Sensitivity of peptide-specific reporter cells. MCR2(gp61)+H18.3.13cells were diluted in MCR2(OVA)+H18.3.13 cells and NFAT activation wasmeasured in untreated cells (triangles) or after co-cultured with SmartaCD4+T-cells(squares). The graph shows a linear (R>0.99) correlationbetween the percentage of GFP+H18.3.13 cells and the percentage of cellscarrying the MCR2(gp61) (f) Minimal frequency of peptide-specificT-cells able to trigger robust NFAT activation in MCR2+ reporter cells.Splenocytes from Smarta and OT-II transgenic mice were mixed atdifferent ratios and used to stimulate MCR2(gp61)+ or MCR2(OVA)+H18.3.13cells. The graph shows percentage of GFP+ cells amongMCR2(gp61)+H18.3.13 or MCR2(OVA)+H18.3.13 cells as a function of thepercentage of peptide-specific CD4+T-cells, after 8 h of co-culture. (g)The minimal number of peptide-specific T cells able to trigger aresponse in reporter cells. NFAT activation in MCR2(gp61)+(left) andMCR2(OVA)+(right) H18.3.13 cells following overnight co-culture withdifferent numbers of Smarta or OT-II CD4+T-cells. h) Kinetics ofLCMV-specific CD4+T-cell expansion in the blood of infected mice (n=3)detected by the activation of MCR2(gp61)+H18.3.13 cells. Percentage ofGFP+ reporter cells after overnight co-culture with blood taken atdifferent days post infection is shown. Blood from a naïve Smarta mousewas used as a positive control.

FIG. 2 shows screening for gp61 mimotopes. (a) A scheme of RAG-mediatedpeptide randomization based on which the MCR2(gp61-RSS) mimotope librarywas generated (see materials and methods), RSS—recombination signalsequence. (b) Examples of altered gp61 sequences found in the library(new amino acids are shown in grey). (c) 16.2c11 cells carrying theNFAT-FT reporter were transduced with the MCR2(gp61-RSS) library or withMCR2(OVA) or MCR2(gp61) as controls and co-cultured with Smarta or OT-IICD4+T-cells hybridomas. Single MCR2(gp61-RSS)+ cells showing NFATactivation (blue-FT fluorescence) after 9 h co-culture with thegp61-specific Smarta hybridoma(top right panel) were sorted andexpanded. (d) Activation of MCR2(gp61)+, MCR2(gp61S)+ andMCR2(gp61N)+16.2c11 cells co-cultured with the Smarta hybridoma. (e)Sequences of the original gp61 and two new gp61 mimotopes found in theMCR2(gp61-RSS) library. (f) CFSE dilution in Smarta CD4+T-cells after a3-day co-culture with dendritic cells pulsed with gp61 mimotopes atdifferent concentrations (histograms) and cytokine production at aconcentration of 100 nM (dot plots).

FIG. 3 shows search for new LCMV epitopes. (a, b) CD4+ T-cells from miceinfected with LCMV were purified from the spleens 5 and 8 days p.i. andfused with BW5147 cells carrying an NFAT-GFP reporter. Reactivity of theresulting hybridomas was determined by GFP expression after co-culturewith LCMV- or gp61-pulsed dendritic cells. Histograms (a) show examplesof different types of reactivity (LCMV-open or gp61-filled dark grey)and the table (b) a summary of the data (c) A scheme of the cloningstrategy for the construction of libraries enriched for naturallyoccurring peptides (NPLs). ORF—open reading frame dictating the naturalprotein sequence (d) MCR2(LCMV-NPL)+16.2c11 reporter cells wereco-cultured with LCMV-reactive hybridomas (H2, H3, H14 and H30) ofunknown peptide-specificity. Activated reporter cells were sorted,expanded and co-cultured again with the corresponding hybridomas. Toachieve enough enrichment this procedure was repeated three times. Dotplots show example NFAT-activation after two or three rounds ofco-culture. (e) After the 3rd round of enrichment, single cells weresorted, expanded and their reactivity verified by co-culture withhybridomas (histogram shows example NFAT reactivity). The table on theright shows frequencies of reactive clones. Peptide sequences from theMCR2 constructs, representing the dominant NP311 epitope and the newNP547 epitope are shown below.

FIG. 4 shows a function test of the MCR1 (chimeric antigen-receptorpolypeptide heterodimer with extracellular parts from MHC I; gp33/H2-Kb)molecule transduced into the H18.3.13 reporter cell line. (A) After anover-night culture in wells coated with αMHC-I antibodies NFATactivation was induced in the MCR1-transduced, but not in control cells.

FIG. 5 shows a) Activation of MCR2(LCMV-NPL)+ 16.2c11 cells after one,two and three rounds of enrichment by co-culture with hybridomas H3 andH4. b) Examples of activation of MCR2+ 16.2c11 clones derived from thescreening of a random peptide MCR2 library with the hybridoma H9 afterthe final co-culture. Examples of sequences recovered from the librarybefore and after enrichment for H9-reactive peptides are shown.

FIG. 6 shows peptide-specific reactivity and sensitivity of the MCR2sensor. a) Time course of peptide-specific NFAT activation (GFP reporterexpression) in MCR2(OVA)+H18.3.13 cells co-cultured with OT-IICD4+T-cells or Smarta CD4+T-cells as controls. Histogram shows examplesof NFAT-activation measurements in MCR2(OVA)+ cells at indicated timepoints b) Sensitivity of peptide-specific reporter cells.MCR2(OVA)+H18.3.13 cells were diluted in MCR2(gp61)+H18.3.13 cells andNFAT activation was measured after co-culture with OT-II CD4+T-cells.The graph shows a linear (R>0.99) correlation between the percentage ofGFP+H18.3.13 cells and the percentage of cells carrying the MCR2(OVA).

EXAMPLES Example 1: Screening of T-Cell Epitopes in Mammalian CellsUsing the Disclosed Chimeric Antigen-Receptor Polypeptide Heterodimer

Current methods to identify cognate T-cell epitopes are based inprinciple on two major approaches. The first approach relies ondetecting physical MHC-TCR interactions by staining T-cells withMHC-tetramers or by staining phage, yeast or insect cells displayingpeptide-MHC complexes with recombinant TCRs. The second approach relieson measuring T-cell activation in co-cultures with dendritic cells (DCs)presenting peptide pools or positional scanning combinatorial peptidelibraries. Screening of MHC-tetramer libraries is effective for definingthe fine-specificity of recognition of known or predicted antigens, butbecause not all peptide-MHC tetramers bind with equal strength, lowaffinity interactions may be easily missed (e.g. 400 times moreOVA-I-A^(b) tetramers than gp66-I-A^(b) tetramers are needed, forsimilar staining of OVA-specific OT-II and gp61-specifc Smarta2 T-cells,respectively). Similar affinity constraints apply to current peptide-MHCdisplay methods, were soluble TCRs are used. Furthermore, MHC moleculeshave to be mutagenized to allow efficient surface expression on phagesor yeast cells. Screening of positional scanning combinatorial peptidelibraries takes advantage of the cross-reactivity of the TCR and usespeptide pools to define motifs that lead to T-cell activation. WhileT-cell epitopes resembling naturally occurring peptides have been foundwith this method, the identified peptides often have no clear homologyto known proteins and one need to resort to bioinformatics approaches.

The inventors disclose herein the development of an universal systemthat allows direct, unbiased, sensitive and efficient epitope screeningin mammalian cells. Such a method should: i) provide a complex mixtureof APCs, each presenting peptides of one, unique, naturally occurringsequence; ii) provide efficient means to identify and separate APCspresenting cognate peptides; iii) offer a possibility to iterativelyrepeat the procedure and iv) allow easy recovery of peptide sequences bycloning. To generate APCs fulfilling the first criteria, the inventorsfollowed the approaches used to produce “single-peptide” mice and toconstruct different peptide-MHC display systems. By means of recombinantDNA technology a peptide was attached directly to the MHC molecule,making a stable complex and preventing other peptides from binding. Alibrary of such peptide-MHC complexes transfected into MHC-deficientcells yields a pool of cells each presenting a unique peptide (fordetails see Materials and Methods). Ideally, identification of APCscarrying cognate peptides for particular T-cells would involve an easilymeasurable signal once their peptide-MHC complexes were bound by theTCRs of the specific T-cells. Therefore the peptide-MHC fusion moleculewas linked to the TCR complex, which is tailor-made for sensinglow-affinity interactions. Direct zeta chain (CD247) fusions have beensuccessfully used to construct various chimeric antigen receptors.However, to create a molecular sensor resembling the native TCR complexas close as possible, the peptide MHC complexes were fused to truncatedTCRα and TCRβ chains consisting of the hinge region, trans-membrane (TM)and intracellular (IC) domains. Connecting the peptide-MHC to the wholeTCR signaling machinery provides more physiological signals. ThisMHC-TCR chimera is referred to as the MCR in the context of thisspecification. Such a molecule, upon transfection into TCR-deficientT-cell hybridomas, allows direct monitoring of peptide-MHC engagement bythe TCRs of specific T-cells using an NFAT-EGFP reporter system (FIG. 1a). Co-culture of cells carrying a library of peptide-MCR molecules withantigen-specific T cell clones or hybridomas allows directidentification of cognate peptide specificities of T-cells by massivelyparallel, functional screening in mammalian cells.

Therefore the MCR was designed and cloned for the screening of cognatepeptides of MHC class II-restricted T-cells, hence MCR2. MCR2 consistsof two chains: the α-chain, composed of the extracellular domains of theI-A^(b) MHC class II α-chain linked to a truncated TCRα; and the β-chaincomposed of a peptide (the dominant LCMV-derived epitope, gp61) and theextracellular domains of the I-A^(b) MHC class II β-chain linked to atruncated TCRβ (FIG. 1a ). A second MCR2 was also cloned carrying theOVA-peptide and the two were designated MCR2(gp61) and MCR2(OVA),respectively. After transduction of the MCRs into a MHC-II⁻TCR⁻ BEKOthymoma cell line, their expression was verified by staining withanti-MHC-II antibodies. As depicted in FIG. 1b , the MCR2 wasefficiently expressed on the cell surface, indicating that it assembledwith CD3 components of the TCR complex. To verify its specificity, BEKOcells expressing MCR2(gp61) or MCR2(OVA) were co-cultured, with purifiedSmarta2 or OT-II CD4⁺ T-cells. A very fast, peptide-specific MCR2down-regulation from the surface was observed, with kinetics identicalto conventional TCRs. MCR2(gp61) was down-regulated in co-cultures withSmarta2 T-cells and not in the presence of OT-II T-cells (FIG. 1c , leftpanel). The reverse was true for the MCR2(OVA), highlighting thespecificity of the MCR2 system (FIG. 1c , right panel). We furtherassessed the ability of the MCR2 to trigger NFAT activation bytransducing it into a TCR-deficient T-cell hybridoma carrying theNFAT-EGFP reporter (H18.3.13). Again, NFAT response was only triggeredwhen MCR2-carrying hybridomas were co-cultured with peptide-specificT-cells (FIG. 1d ). The response was robust and easily measurablealready after 2 h (FIG. 1d most right panel).

The inventors tested the sensitivity of the MCR system by mixingMCR2(gp61)⁺ and MCR2(OVA)⁺ reporter cells at different ratios andmeasuring NFAT-activation after co-culture with Smarta2 or OT-II CD4⁺T-cells. As shown in FIG. 1e , it was possible to directly detectspecific NFAT-reporter expression in cells present at frequencies above1/10000. Importantly, a linear correlation between the percentage ofdetected NFAT-EGFP expressing cells and the percentage of cells carryingthe “T-cell/idotype-specific” MCR2 was observed, indicating thatspecific cells present at frequencies lower than 1/10000 are stillNFAT-EGFP′, even if they cannot be distinguished from the background.The lowest frequency of peptide specific T-cells in a heterogeneouspopulation, that was able to trigger robust NFAT-activation in MCR2⁺cells was also determined. Sorted CD4⁺ T-cells (FIG. 1f , top panel) orunsorted splenocytes (FIG. 1f , bottom panel) from Smarta2 and OT-IImice were mixed at different ratios and used to stimulate MCR2(gp61)⁺ orMCR2(OVA)⁺ cells. Even with only 1% peptide-specific CD4⁺ T-cells, 50%of the maximal NFAT-activation was triggered in MCR2⁺ cells, whenT-cells were provided in excess. Remarkably, as shown in FIG. 1g (leftpanel), even a single Smarta2 CD4⁺ T-cell was able to triggersignificant NFAT-EGFP activation in MCR2(gp61)⁺ cells, while in the caseof MCR2(OVA)⁺ cells, 5 cells were required, probably due to a lowerinteraction affinity (FIG. 1g right panel). These results indicate thatthe MCR-technology can be used as a sensitive diagnostic tool formonitoring T cell specificities in the blood taken from patients. IndeedMCR2(gp61)⁺ reporter cells can be used to efficiently trackantigen-specific CD4⁺ T-cell expansion in the blood of LCMV-infectedanimals (FIG. 1h ). These results demonstrate the great sensitivity ofthe MCR-method.

To use the disclosed invention for finding rare specific peptides in acomplex library, multiple iterative cycles of co-culture and sorting ofNFAT-EGFP⁺ reporter cells are necessary. Because efficient detection ofNFAT-activation in subsequent rounds of stimulation depends on fastdisappearance of NFAT-reporter signals triggered in previous rounds, thevery stable EGFP was replaced with the slow Fluorescent Timer (sFT).This mutant of mCherry changes “color” with time, enabling thedistinction of recent (blue-mCherry) and past NFAT-activation(red-mCherry) and therefore allows for much shorter intervals betweensubsequent rounds of stimulation.

First the disclosed invention was applied to search for mimotopes ofgp61 in the MCR2(gp61-RSS) library, generated by randomizing centerresidues of gp61 through RAG-mediated rearrangement (FIG. 2a and M&M).Randomly picked clones consistently contained unique mutants of thegp61-sequence (FIG. 2a ). After transducing this library into NFAT-sFTcarrying reporter cells (16.2c11), MCR2⁺ cells were sorted, expanded andco-cultured with gp61-specific or OVA-specific T-cell hybridomas (FIG.2b ). Around 10% of the MCR2(gp61-RSS/I-A^(b))⁺16.2 c11 cells showedblue NFAT-reporter activation when co-cultured with gp61-specifichybridomas. These cells were sorted as single cells, expanded andrescreened (FIG. 2c ). All of the 24 tested clones responded tore-stimulation with gp61-specific hybridomas. PCR-amplification andsequencing of the peptide parts of the MCRs from these clones revealedtwo new mimotopes and the original gp61 peptide (FIG. 2c ). The lysineat position 9 of gp61 was mutated to serine (gp61S) or to asparagine(gp61N), indicating that it is not absolutely required for Smarta2T-cell activation. However, the level of NFAT-activation suggested thatSmarta2 TCR binds the new mimotopes (in particular gp61S) with loweraffinity (histogram in FIG. 2c ). In co-cultures of T-cells withdendritic cells, both mimotopes induced robust Smarta2 T-cell responses.Interestingly, while gp61N induced proliferation and Th1-like cytokineproduction similarly to the original peptide, the suboptimal gp61S wasmuch less efficient in driving proliferation, but induced a strongTh2-like cytokine response (FIG. 2d ).

Finally, a screen for novel LCMV epitopes with the help of CD4⁺ T-cellhybridomas derived from LCMV-infected animals 5 and 8 days postinfection was performed (FIGS. 3a and b ). The hybridomas carried anNFAT-EGFP reporter that allowed verification of their reactivity againstLCMV and pick gp61-nonreactive hybridomas for further analysis (FIG. 3b). Five such hybridomas (H30,H14,H2 responding strongly and H4,H3responding weekly) were used to screen 16.2c11 reporter cells transducedwith a library of MCR2 molecules carrying all possible, overlappingpeptides of the LCMV glycoprotein (GP) and nuclear protein (NP). This“genuine peptide library” (GPL) was generated by cloning random piecesof cDNA encoding GP and NP into the MCR2 vector (FIG. 3c ) and has asignificant advantage over random or combinatorial peptide libraries, asmany (⅙) of the recovered peptides represent native proteins. Indeed,for 4 out of 5 hybridomas, the LCMV-specific target peptides weredirectly identified. Three of the hybridomas (H30,H14 and H2) recognizeda known dominant LCMV-epitope NP311 and H3 reacted with a new epitopeNP547 (FIG. 3d ). The fifth hybridoma (H4) did not yield any enrichmentfor reactive reporter cells even after 3 iterative rounds of screening.TCR surface expression was tested on this hybridoma and was found to beTCR-negative. The TCR was probably lost during expansion after theinitial LCMV-specificity screening. This result further verifies theTCR-specificity of the disclosed method. One additional hybridoma (H9)was used to screen a MCR2 library carrying random peptides and foundseveral strongly reactive epitopes, but they did not resemble any LCMVpeptides. This further supports the strategy of using GPLs rather thanrandom peptide libraries for T-cell epitope screening.

Herein a new molecular sensor is disclosed, which allows for sensing ofpeptide-MHC-TCR interactions on the APC side with great specificity,sensitivity and fast kinetics. Using this reporter, a novel approach forunbiased, functional screening of T-cell epitopes was established. Itcombines the versatility of expression cloning with the sensitivity andhigh-throughput capabilities of fluorescence activated cell sorting andallows for efficient iterative screening of peptide libraries inmammalian cells. All this provides significant advantages over themethods known in the art. First, thanks to the multivalent interactionbetween the MCRs and TCRs, high and low affinity binding generatesimilar NFAT-reporter signals (FIG. 1d ) and therefore low affinityligands are less likely to be missed. Indeed, even though LCMV epitopeshave been extensively studied, a novel epitope—NP547 could beidentified. Second, engineering of individual recombinant TCRs ormutagenesis of the MHC are not required, as the peptides are screened inthe context of native TCR and MHC molecules expressed on the surface ofmammalian cells. Third, as exemplified by the LCMV virus peptide screen,the MCR-technology facilitates efficient screening of libraries highlyenriched for peptides derived from the pathogen/cell/tissue targeted bythe T-cell of interest.

The MCR-based approach provides a versatile, easy to use and powerfulway of identifying antigenic specificities of T-cells. As such, it mayimpact several fields of basic and clinical research. Definingspecificities of regulatory and effector tumour-infiltrating T-cellsenables the discovery of novel tumour-antigens. Defining thespecificities of auto-reactive tissue-infiltrating T-cells aids in thedevelopment of antigen-specific therapies for autoimmune diseases. Inthis respect, MCR may also allow for efficient redirecting of T-celleffector functions towards peptide-specific T-cells, enabling thepurging of the repertoire from undesired specificities. Furthermore,screening of mimotope libraries will lead to the discovery of highaffinity peptide variants and the development of sensitive flowcytometry based tests for antigenic reactivity of T-cells circulating inthe blood of patients.

Materials and Methods

Mice

C57/Bl6, mice were purchased from Charles River. Smarta2 and OT-II micewere bred at the ETH mouse facility.

Cell Lines

Beko is a spontaneous thymoma cell line derived from TCRβ-deficientmice. The H18.3.13 reporter cell line was generated by retrovirallytransducing the NFAT-EGFP reporter (carrying four copies of the minimalhuman IL-2 promoter, each containing 3 NFAT binding sitesACGCCTTCTGTATGAAACAGTTTTTCCTCC (SEQ ID NO 001), inserted upstream of theEGFP coding sequence) into a TCR⁻ B6 T-cell hybridoma. The 16.2c11reporter cell line was generated by transfecting the 16.2 T-cellhybridoma with the NFAT-sFT reporter construct and a vector encoding themurine eco-tropic retrovirus receptor Slc7a1.

Hybridoma generation Sorted T-cells or thymocytes were activated withplastic-bound anti-CD3E and anti-CD28 antibodies in the presence ofmouse IL-2 for 2-3 days. Equal numbers of activated T-cells and theTCRα⁻β⁻ BW5147 fusion partner were fused using PEG-1500, and plated atlimiting dilution in the presence of 100 mM hypoxanthine, 400 nMaminopterin, and 16 mM thymidine (HAT).

Cloning of the MCR2

The MCR2 α and β chains were cloned by standard techniques and containthe following parts:

MCR2 α chain: the MHC-II I-A^(b) α chain residues 1-208 linked to theTCRα chain constant region residues 87-137 by the GGSGGSAQ (SEQ ID NO002) linker.

MCR2 β chain: the MHC-II I-A^(b)β chain residues 1-217 linked to theTCRβ chain constant region (C1) residues 123-173 by the AQSGGSGGSAQ (SEQID NO 003) linker. In the MCR2(gp61) residues DS at positions 29 and 30of the MHC-II part were replaced by the amino acid sequence

SGLNGPDIYKGVYQFKSVGSGGSGGSGDS (SEQ ID NO 004;containing the gp61 peptide).In MCR2(OVA) the same residues were replaced by the amino acid sequence

SISQAVHAAHAEINEAGRGSGGSGGSGDS (SEQ ID NO 005;containing the OVA peptide).

Retro Viral Transduction of Reporter Cell Lines and Sorted Thymocytes

MCRα and MCRβ were cloned into the pMYiresGFP retroviral vector, so thatMCRβ replaced GFP. Throughout the study we used this vector(pMY-MCRαiresMCRβ) to generate MCRs containing various peptides andreferred to them as MCR(“peptide”/“MHC haplotype”). Retroviruscontaining supernatants were produced in the ecotropic Phoenix packagingcell line and used to infect reporter cell lines and sorted cells.

RAG-Mediated Generation of Mimotope Libraries

To generate the gp61 mimotope library, theMCR2(gp61-RSS-EGFP-RSS/I-A^(b)) construct was built by inserting astuffer fragment containing EGFP and the RAG recombination signalsequences (RSS) into the middle of the gp61 peptide in the MCR2(gp61)construct (FIG. 2a ). This construct was transduced into sorted CD4⁻CD8⁻double-negative (DN) thymocytes cultured on Tst-4/DLL1³². After a weekof culture (during which DN cells develop into CD4⁺CD8⁺ double-positive(DP) cells and recombine their TCR genes, as well as the RSS-EGFP-RSSstuffer, by RAG-mediated rearrangement) cells expressing low levels ofEGFP were sorted and cDNA was made. The peptide-encoding part of therecombined MCR2(gp61-RSS-EGFP-RSS/I-A^(b)) construct was PCR amplifiedfrom the cDNA and cloned into the empty MCR2(I-A^(b)) vector, generatingthe MCR2(gp61-RSS/I-A^(b)) mimotope library.

Genuine and Random Peptide Library Generation and Screening

To generate the MCR2-LCMV genuine overlapping peptide library DNAencoding the GP and NP proteins was digested for a limited amount oftime (Takara DNA fragmentation Kit). The fragments were ligated withlinkers homologous to vector sequences flanking the cloning site, PCRamplified, cloned into the pMY-MCR2 vector by Gibsson assembly andtransfected into bacteria generating over 2*10⁶ clones. 16.2c11 cellswere transduced with this library and 0.5*10⁶ MCR^(low) and 2.2*10⁴MCR^(hi) cells were sorted.

The MCR2 random peptide library was made by cloning an oligonucleotide(GGTNNNNNNTWCNNNNNNBCCNNNSCCNNNNNNKCCNNNGGA) (SEQ ID NO 006) into theMCR2-vector using the strategy described above. This oligonucleotideencoded random amino acids at positions facing the TCR, while anchorresidues were partially fixed to ensure good presentation. Thecomplexity was 5.5*10⁶ bacterial clones and after transduction 11.5*10⁶individual MCR2+ cells were sorted.

MCR Down-Regulation Assay

If not stated otherwise, MCR2⁺ Beko cells were co-cultured with a 5-foldexcess of sorted CD4⁺ T-cells from indicated donor mice.

Stimulation of MCR⁺ H18.3.13 or 16.2c11 Cells

If not stated otherwise, MCR2⁺ cells were co-cultured with a 5-foldexcess of sorted CD4⁺ T-cells or CD4⁺ T-cell hybridomas from indicateddonor mice for 8-12 h.

Example 2: Chimeric Antigen-Receptor Polypeptide Heterodimer

First polypeptide, alpha chain (SEQ ID NO 007):MPRSRALILGVLALTTMLSLCGGEDDIEADHVGTYGISVYQSPGDIGQYTFEFDGDELFYVDLDKKETVWMLPEFGQLASFDPQGGLQNIAVVKHNLGVLTKRSNSTPATNEAPQATVFPKSPVLLGQPNTLICFVDNIFPPVINITWLRNSKSVADGVYETSFFVNRDYSFHKLSYLTFIPSDDDIYDCKVEHWGLEEPVLKHWEPEGGSGGSAQSDVPCDATLTEKSFETDMNLNFQNLSVMG LRILLLKVAGFNLLMTLRLWSS

Amino acids 1-208 are derived from MHC2 alpha. Amino acids 209-216 are alinker sequence. Amino acids 217-267 are derived from TCR alpha.

Second polypeptide, beta chain (SEQ ID NO 008):MALQIPSLLLSAAVVVLMVLSSPRTEGGSGGSGGSGDSERHFVYQFMGECYFTNGTQRIRYVTRYIYNREEYVRYDSDVGEHRAVTELGRPDAEYWNSQPEILERTRAELDTVCRHNYEGPETHTSLRRLEQPNVVISLSRTEALNHHNTLVCSVTDFYPAKIKVRWFRNGQEETVGVSSTQLIRNGDWTFQVLVMLEMTPRRGEVYTCHVEHPSLKSPITVEWRAQSGGSGGSAQGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS

Amino acids 1 to 26 are a leader peptide. Between amino acids 27 and 28is the insertion site of oligopeptides to be displayed. Amino acids 29to 36 are a linker sequence. Amino acids 37 to 228 are derived from MHC2beta. Amino acids 229 to 236 are a linker sequence. Amino acids 237 to287 are derived from TCRbeta.

Peptides to be inserted in SEQ ID NO 008 between amino acids 27 and 28(GG):

Gp61 (SEQ ID NO 009): LNGPDIYKGVYQFKSV OVA (SEQ ID NO 010):ISQAVHAAHAEINEAGR

1-13. (canceled)
 14. A method for the identification of a TCRrecognizable peptide sequence, comprising: i. providing a plurality ofmammalian cells, wherein each of said plurality of mammalian cellsexpresses a member of a library; each member of said library encodes atransgenic antigen receptor; said transgenic antigen receptor comprises:an extracellular domain of MHC1 or MHC2, an oligopeptide comprisedwithin said extracellular domain, wherein the oligopeptide is differentfor each member of the library, and wherein the oligopeptide ispresented in a way suitable for the recognition by a T cell receptor, atransmembrane domain of a T-cell receptor (TCR), and an intracellulardomain of a TCR; said transgenic antigen receptor is functionally linkedto a reporter protein, whereby binding of a cognate T cell receptor tosaid transgenic antigen receptor results in the activation of saidreporter gene, ii. contacting said plurality of mammalian cells with apreparation of T-lymphocytes, iii. separating cells showing a detectablereporter protein from said plurality of mammalian cells according to thedetectable level of said reporter protein, yielding activated cells, iv.isolating DNA from said activated cells, and v. sequencing of saidoligopeptide sequence comprised in said transgenic antigen-receptor. 15.A method for the identification of a TCR recognizable peptide sequence,comprising: i. providing a mammalian cell, wherein said mammalian cellexpresses a transgenic antigen receptor; said transgenic antigenreceptor molecule comprises an extracellular domain of MHC1 or MHC2comprising an oligopeptide presented in a way suitable for therecognition by a T cell receptor, a transmembrane domain of a T-cellreceptor (TCR), and an intracellular domain of a TCR, said transgenicantigen receptor is functionally linked to a reporter protein, wherebybinding of a cognate T cell receptor to said transgenic antigen receptorresults in the activation of said reporter gene, ii. contacting saidmammalian cell with a preparation of T-lymphocytes, thereby activatingsaid reporter gene yielding an activated mammalian cell, iii. isolatingDNA from said activated mammalian cell, and iv. sequencing of saidoligopeptide sequence encoded in said transgenic chimericantigen-receptor.
 16. The method according to claim 14 or 15, whereinsaid transgenic antigen receptor is a chimeric antigen-receptorpolypeptide heterodimer comprising a first polypeptide and a secondpolypeptide, wherein a. said first polypeptide comprises anextracellular part of the major histocompatibility complex I (MHC classI) alpha chain and said second polypeptide comprises a β2-microglobulindomain, or b. said first polypeptide comprises an extracellular part ofthe major histocompatibility complex II (MHC class II) alpha chain andsaid second polypeptide comprises an extracellular part of the majorhistocompatibility complex II (MHC class II) beta chain, wherein atleast one of said first and said second polypeptide chain additionallycomprises an oligopeptide, covalently linked to said extracellular MHCdomain; one of said first polypeptide and said second polypeptidefurther comprises a hinge region, a transmembrane domain and anintracellular domain or intracellular tail of the T cell receptor alphachain and the other one of said first polypeptide and said secondpolypeptide comprises a hinge region, a transmembrane domain and anintracellular domain of the T cell receptor beta chain.